Natural Product Sciences 19(3) : 227-230 (2013)
227
Iridoid Compounds from the Whole Plant of Galium verum var. asiaticum
Tae Gwan Lee and Dae Keun Kim*
College of Pharmacy, Woosuk University, Jeonju 565-701, Korea
Abstract – One new iridoid glycoside, 10-p-dihydrocoumaroyl-6-α-hydroxygeniposide (1), and six known iridoid glycoside derivatives, 10-p-dihydrocoumaroyl deacetylasperuloside (2), asperulosidic acid methylester (3), asperuloside (4), asperulosidic acid (5), deacetylasperuloside (6), and scandoside (7) were isolated from the methanolic extract of the whole plant of Galium verum var. asiaticum Nakai (Rubiaceae) through repeated column chromatography. Their chemical structures were characterized by spectroscopic analysis. This is the first report of the characterization of compounds 1 - 7 from this plant.
Keywords – Galium verum var. asiaticum, Rubiaceae, Iridoid glycoside
Introduction
Galium verum var. asiaticum Nakai (Rubiaceae) is a perennial plant, widely distributed in Korea (Lee, 2003;
1996). This plant has been used as a traditional medicinal plant for the treatment of hepatitis, tonsillitis and dermatitis in Korea (Kang, 2008). Previous phytochemical investigations of the genus Galium resulted in the isolation of iridoid glycosides, anthraquinones, and triterpenes (Morimoto et al., 2002; de Rosa et al., 2000;
Handjievaetal., 1996; El-Gamal et al., 1995; Uesato et al., 1984; Böjthe-Horváth et al., 1980). From this plant, several phenolic compounds were reported from the n- butanol soluble fraction as antioxidative constituents (Kim, 2011). Except for those phenolic compounds, phytochemical and pharmacological studies of G. verum var. asiaticum have not been performed yet. Therefore phytochemical studies were performed to investigate their components. This paper presents information on the isolation and structure elucidation of compounds 1 - 7.
Experimental
General experimental procedures –1H and 13C NMR spectra were determined on a JEOL JMN-EX 400 spectrometer. UV spectra were recorded on Shimadzu UV-1601 UV-Visible spectrophotometer, and Mass spectra were acquired on a JEOL JMSAX 505-WA. TLC
was carried out on Merck (Darmstadt, Germany) precoated silica gel F254 plates, and silica gel for column chromatography was Kiesel gel 60 (230 - 400 mesh, Merck). Spots were detected under UV and by spraying with 10% H2SO4 in ethanol followed by heating at 100 - 120oC for 3 min. Sephadex LH-20 was used for column chromatography (25 - 100 µm; GE Healthcare, Uppsala, Sweden). Prep-HPLC was carried out on a Jaigel GS310 column (Tokyo, Japan). Methanol-d4 (CD3OD) for NMR experiment was obtained from Merck (Darmstadt, Germany). Solvents used for analyses were of HPLC grade and purchased from Fisher Scientific Korea (Seoul, Korea). Methanol, n-hexane, ethyl acetate, methylene chloride, and n-butanol for extraction and solvent fractionation were purchased from Samchun Chemical (Pyeongtack, Korea).
Plant materials – The whole plant of G. verum var.
asiaticum was collected in August 2011 at Wanju, Jeonbuk, Korea, and identified by Dae Keun Kim, College of pharmacy, Woosuk University. A voucher specimen was deposited in the herbarium of the College of Pharmacy, Woosuk University (WSU-11-040).
Extraction and isolation – The shade dried plant material (550 g) was extracted three times with methanol at 50oC and filtered. The extracts were combined and evaporated in vacuo at 50oC. The resultant methanol extract (43 g) was successively partitioned as n-hexane (5.5 g), methylene chloride (2.0 g), n-BuOH (12.7 g) and H2O soluble fractions. Sephadex LH-20 (MeOH) column chromatography of n-BuOH soluble (3 g) extract gave six fractions (B1-B6). Fraction B1 (1.6 g) was chromato- graphed on silica gel column chromatography (EtOAc-
*Author for correspondence
Dae Keun Kim, College of Pharmacy, Woosuk University, Jeonju 565-701, Korea
Tel: +82-63-290-1574; E-mail: dkkim@woosuk.ac.kr
228 Natural Product Sciences
MeOH-H2O, 50 : 10 : 1) as an eluent to give five subfractions (B11-B15). Subfraction B11 was purified by HPLC column (MeOH) to give compounds 1 (6.8 mg) and 2 (11.2 mg), respectively. Subfraction B12 was purified by HPLC column (MeOH) to give compound 3 (25 mg) and compound 4 (30 mg), respectively. Subfraction B13 was purified by HPLC column (MeOH) to give compounds 5 (7 mg) and 6 (15 mg), respectively.
Subfraction B15 was purified by HPLC column (MeOH) to give compound 7 (8 mg).
10-p-dihydrocoumaroyl-6-α-hydroxygeniposide (1) – Amorphous solid; HR-FABMS 575.1577 [M + Na]+ (C26H32NaO13+, calc. 575.1579); UV λmax (MeOH) nm 225, 315; 1H and 13C NMR Table 1 andTable 3.
10-p-dihydrocoumaroyl deacetylasperuloside (2) – Amorphous solid; UV λmax (MeOH) nm 223, 281; 1H and
13C NMR Table 1 andTable 3.
Asperulosidic acid methylester (3) – Amorphous solid;
UV λmax (MeOH) nm 233; 1H and 13C NMR Table 2 and Table 3.
Asperuloside (4) – Amorphous solid; UV λmax (MeOH) nm 232; 1H and 13C NMR Table 2 andTable 3.
Asperulosidic acid (5) – Amorphous solid; UV λmax
(MeOH) nm 230; 1H and 13C NMR Table 2 andTable 3.
Deacetylasperuloside (6) – Amorphous solid; UV λmax
(MeOH) nm 233; 1H and 13C NMR Table 2 andTable 3.
Scandoside (7) – Amorphous solid; UV λmax (MeOH) nm 235; 1H and 13C NMR Table 2 andTable 3.
Results and Discussion
The methanol extract was suspended in water and partitioned successively with n-hexane, methylene chloride and n-BuOH. After observation of TLC patterns of each fraction, the n-BuOH soluble fraction was subjected to column chromatography. Through several repeated chromatographies using Silica gel, Sephadex LH-20, and HPLC, seven iridoid glycoside derivatives were isolated from this fraction.
Compounds 2, 4, and 6 have similar patterns in their NMR spectra except for the dihydrocoumaroyl group of 2, and the acetyl signal of 4. Compounds 3 and 5 have similar patterns in their NMR spectra except for the methoxyl group of 3. Structure characterization of compounds 2 - 7 was carried out by interpretation of their spectral data and comparison with the data previously reported in the literature. Compounds 2 - 7 were identified as 10-p-dihydrocoumaroyl deacetylasperuloside (2) (Böjthe- Horváth et al., 1982), asperulosidic acid methylester (3) (El-Naggar and Beal, 1980; Takeda et al., 2002), asperuloside (4) (El-Naggar and Beal, 1980; Lee et al., 2004), asperulosidic acid (5) (El-Naggar and Beal, 1980;
Chaudhuri et al., 1980), deacetylasperuloside (6) (Myagoshi et al., 1987; Peng et al., 1997), and scandoside (7) (Chaudhuri et al., 1980), respectively.
Compound 1 was obtained as an amorphous powder, and molecular composition of compound 1 was determined to be C26H32O13 by FABMS. Its UV spectrum showed maximum absorption at 225 nm from an aromatic ring Fig. 1. Structures of isolated compounds.
Table 1. 1H-NMR spectral data of compounds 1 and 2
C 1 2
1 3 5 6 7 9 10
1' 2",6"
3",5"
αβ OCH3
5.03 (1H, d, J = 9.2) 7.64 (1H, s) 4.70 (1H, m) 4.75 (1H, m) 5.88 (1H, s) 2.98 (1H, m) 4.93 (1H, d, J = 15.4) 4.73 (1H, d, J = 15.4) 4.70 (1H, d, J = 8.0) 7.02 (2H, d, J = 8.4) 6.68 (2H, d, J = 8.4) 2.84 (2H, t, J = 7.6) 2.64 (2H, d, J = 7.6) 3.73 (3H, s)
5.96 (1H, d, J = 0.8) 7.26 (1H, d, J = 2.0) 4.70 (1H, m) 5.51 (1H, d, J = 6.4) 5.93 (1H, s) 3.38 (1H, m) 4.74 (1H, d, J = 14.4) 4.62 (1H, d, J = 14.4) 4.66 (1H, d, J = 8.0) 7.01 (2H, d, J = 8.4) 6.68 (2H, d, J = 8.4) 2.82 (2H, t, J = 7.2) 2.63 (2H, d, J = 7.2) Recorded at 400 MHz in CD3OD
Vol. 19, No. 3, 2013 229
conjugated with a ketone function. In the 13C-NMR spectrum of compound 1, an acyl moiety composed of aromatic signals (δ 156.8, 132.6, 130.3, 116.3) and a carbonyl carbon signal (δ 174.5) were observed. Except for the acyl portion of compound 1 and acetyl group of compound 3, NMR chemical shifts of compounds 1 and 3 showed very similar patterns to each other. The characteristic peak of α-configuration of hydroxyl group at C-6 of the iridoid compound was observed at δ 75.4 in the 13C-NMR spectrum of compound 1. In the 1H-NMR spectra, an A2B2 system appears between δ 7.02 and 6.68
(each 2H, d, J = 8.4) of compound 1 indicating a p- disubstituted benzene derivative. In addition, a set of signals intergrating for four hydrogens and characteristic of an A2B2 system appears between δ 2.84 and 2.64 (each 2H, t, J = 7.6). These data implied the acyl portion is a dihydrocoumaroyl. According to the down shifted chemical shift values and signal patterns of the 2H-10 protons, compound 1 is acylated at this point (Böjthe-Horváth et al., 1982), and long-range coupling signal between carbonyl carbon of dihydrocoumaroyl group and H-10 protons was observed in the HMBC NMR spectrum of Table 2. 1H-NMR spectral data of compounds 3 - 7
C 3 4 5 6 7
1 3 5 6 7 9 10
1' OCH3
CH3
5.05 (1H, d, J = 8.8) 7.65 (1H, s) 2.63 (1H, t, J = 8.4) 4.76 (1H, m) 6.02 (1H, s) 3.03 (1H, m) 4.89 (1H, d, J = 16.0) 4.75 (1H, d, J = 16.0) 4.71 (1H, d, J = 7.6) 3.74 (3H, s) 2.08 (3H, s)
5.96 (1H, br.d, J = 0.8) 7.30 (1H, d, J = 2.4) 3.75 (1H, m)
5.57 (1H, br.d, J = 6.4) 5.73 (1H, s)
3.38 (1H, m) 4.78 (1H, d, J = 14.4) 4.66 (1H, d, J = 14.4) 4.67 (1H, d, J = 8.0) 2.08 (3H, s)
5.05 (1H, d, J = 8.8) 7.59 (1H, s) 3.02 (1H, m) 4.82 (1H, br. s) 6.01 (1H, s) 2.61 (1H, m) 4.93 (1H, d, J = 15.5) 4.80 (1H, d, J = 15.5) 4.72 (1H, d, J = 8.0) 2.08 (3H, s)
5.94 (1H, br.d, J = 0.8) 7.28 (1H, d, J = 2.0) 3.65 (1H, m)
5.55 (1H, br.d, J = 6.8) 5.63 (1H, s)
3.37 (1H, m) 4.18 (2H, s) 4.67 (1H, d, J = 7.6)
4.85 (1H, d, J = 7.2) 7.33 (1H, s) 3.65 (1H, m) 4.53 (1H, m) 5.82 (1H, s) 2.89 (1H, m) 4.36 (1H, d, J = 15.5) 4.17 (1H, d, J = 15.5) 4.71 (1H, d, J = 8.4)
Recorded at 400 MHz in CD3OD
Table 3. 13C-NMR spectral data of compounds 1 - 7
C 1 2 3 4 5 6 7
1 3 4 5 6 7 8 9 10 11 1' 2' 3' 4' 5' 6'α β 1"
2"
3"
4"
5"
6"
C=O OCH3
CH3
100.7 155.4 108.1 42.4 75.4 131.9 145.9 46.2 63.6 169.4 101.4 74.9 78.6 71.6 77.9 63.0 37.1 31.2 132.6 130.3 116.3 156.8 116.3 130.3 174.5 51.8
100.0 150.3 106.1 37.0 86.3 129.1 144.2 45.1 62.8 172.6 93.3 74.7 78.3 71.6 77.9 61.7 37.4 31.0 129.1 130.4 116.3 156.8 116.3 130.4 174.2
100.6 155.4 108.1 42.4 75.4 131.8 146.0 46.3 63.8 169.3 101.3 74.9 78.6 71.6 77.9 63.0
172.5 51.8 20.7
100.0 150.3 106.1 37.4 86.3 128.9 144.2 45.2 62.8 172.3 93.3 74.6 78.3 71.6 77.8 61.9
172.6 20.6
101.1 154.4 109.8 42.8 75.5 131.9 146.0 46.4 63.8 172.5 100.5 75.0 78.6 71.6 77.9 63.0
172.6 20.7
99.9 150.3 106.5 37.5 86.7 125.7 149.8 45.0 62.8 172.9 93.3 74.7 78.4 71.6 77.9 60.1
99.4 150.3 116.0 47.3 83.1 129.9 147.4 48.3 61.6 173.1 100.3 75.0 78.4 71.5 77.8 62.6
Recorded at 100 MHz in CD3OD
230 Natural Product Sciences
compound 1. The characteristic peak of α-configuration of hydroxyl group at C-6 was observed at δ 75.4 in the
13C-NMR spectrum of compound 1 (β configuration of hydroxyl group is usually observed around at δ 83.0).
NMR spectra indicate that compound 1 is a 10-O- acylated derivative of asperulosidic acid methyl ester. On the basis of the above evidences, the structure of compound 1 was determined to be 10-p-dihydrocoumaroyl- 6-α-hydroxygeniposide.
To our best knowledge, this is the first report on the elucidation of compound 1 in the nature, and compounds 2-7 were characterized for the first time from this plant.
Acknowledgements
This work was supported by the Leaders in INdustry- university Cooperation (LINC) of Woosuk University.
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Received May 7, 2013 Revised June 12, 2013 Accepted June 14, 2013
